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Robustness of hen lysozyme monitored by random mutations
Kaori Kunichika1, Yoshio Hashimoto, Taiji Imoto
1Graduate School of Pharmaceutical Science, Kyushu University, Fukuoka 812-8582, Japan.
Protein Engineering
|December 7, 2002
Summary
Hen lysozyme
Area of Science:
- Protein Engineering
- Biochemistry
- Molecular Biology
Background:
- Hen lysozyme is a model protein for studying protein stability.
- Understanding protein robustness is crucial for protein design and function.
Purpose of the Study:
- To investigate the impact of random mutations on hen lysozyme's structure and activity.
- To determine the robustness of hen lysozyme's gross conformation and active structure.
Main Methods:
- Construction of six random mutant libraries of hen lysozyme with varying mutation levels (1-14 amino acids).
- Systematic variation of Mg(2+) and Mn(2+) concentrations during polymerase chain reaction.
- Screening of 4000 clones using lysis activity assays and ELISA with a conformation-specific monoclonal antibody.
Main Results:
- Approximately 80% of clones with an average of two mutations retained active structure.
- Nearly all clones with an average of five mutations lost active structure.
- 80% of clones with an average of two mutations maintained gross conformation, while 24% with 14 mutations retained gross conformation.
Conclusions:
- Hen lysozyme's gross conformation is highly robust against mutations.
- The active structure of hen lysozyme is also robust, but to a lesser extent than its gross conformation.